The voltage-dependent potassium channel subunit Kv2.1 regulates insulin secretion from rodent and human islets independently of its electrical function.

AIMS/HYPOTHESIS: It is thought that the voltage-dependent potassium channel subunit Kv2.1 (Kv2.1) regulates insulin secretion by controlling beta cell electrical excitability. However, this role of Kv2.1 in human insulin secretion has been questioned. Interestingly, Kv2.1 can also regulate exocytosis through direct interaction of its C-terminus with the soluble NSF attachment receptor (SNARE) protein, syntaxin 1A. We hypothesised that this interaction mediates insulin secretion independently of Kv2.1 electrical function. METHODS: Wild-type Kv2.1 or mutants lacking electrical function and syntaxin 1A binding were studied in rodent and human beta cells, and in INS-1 cells. Small intracellular fragments of the channel were used to disrupt native Kv2.1-syntaxin 1A complexes. Single-cell exocytosis and ion channel currents were monitored by patch-clamp electrophysiology. Interaction between Kv2.1, syntaxin 1A and other SNARE proteins was probed by immunoprecipitation. Whole-islet Ca(2+)-responses were monitored by ratiometric Fura red fluorescence and insulin secretion was measured. RESULTS: Upregulation of Kv2.1 directly augmented beta cell exocytosis. This happened independently of channel electrical function, but was dependent on the Kv2.1 C-terminal syntaxin 1A-binding domain. Intracellular fragments of the Kv2.1 C-terminus disrupted native Kv2.1-syntaxin 1A interaction and impaired glucose-stimulated insulin secretion. This was not due to altered ion channel activity or impaired Ca(2+)-responses to glucose, but to reduced SNARE complex formation and Ca(2+)-dependent exocytosis. CONCLUSIONS/INTERPRETATION: Direct interaction between syntaxin 1A and the Kv2.1 C-terminus is required for efficient insulin exocytosis and glucose-stimulated insulin secretion. This demonstrates that native Kv2.1-syntaxin 1A interaction plays a key role in human insulin secretion, which is separate from the channel's electrical function.

Direct interaction of a brain voltage-gated K+ channel with syntaxin 1A: functional impact on channel gating.

Presynaptic voltage-gated K(+) (Kv) channels play a physiological role in the regulation of transmitter release by virtue of their ability to shape presynaptic action potentials. However, the possibility of a direct interaction of these channels with the exocytotic apparatus has never been examined. We report the existence of a physical interaction in brain synaptosomes between Kvalpha1.1 and Kvbeta subunits with syntaxin 1A, occurring, at least partially, within the context of a macromolecular complex containing syntaxin, synaptotagmin, and SNAP-25. The interaction was altered after stimulation of neurotransmitter release. The interaction with syntaxin was further characterized in Xenopus oocytes by both overexpression and antisense knock-down of syntaxin. Direct physical interaction of syntaxin with the channel protein resulted in an increase in the extent of fast inactivation of the Kv1.1/Kvbeta1.1 channel. Syntaxin also affected the channel amplitude in a biphasic manner, depending on its concentration. At low syntaxin concentrations there was a significant increase in amplitudes, with no detectable change in cell-surface channel expression. At higher concentrations, however, the amplitudes decreased, probably because of a concomitant decrease in cell-surface channel expression, consistent with the role of syntaxin in regulation of vesicle trafficking. The observed physical and functional interactions between syntaxin 1A and a Kv channel may play a role in synaptic efficacy and neuronal excitability.

Fast inactivation of a brain K+ channel composed of Kv1.1 and Kvbeta1.1 subunits modulated by G protein beta gamma subunits.

Modulation of A-type voltage-gated K+ channels can produce plastic changes in neuronal signaling. It was shown that the delayed-rectifier Kv1.1 channel can be converted to A-type upon association with Kvbeta1.1 subunits; the conversion is only partial and is modulated by phosphorylation and microfilaments. Here we show that, in Xenopus oocytes, expression of Gbeta1gamma2 subunits concomitantly with the channel (composed of Kv1.1 and Kvbeta1.1 subunits), but not after the channel's expression in the plasma membrane, increases the extent of conversion to A-type. Conversely, scavenging endogenous Gbetagamma by co-expression of the C-terminal fragment of the beta-adrenergic receptor kinase reduces the extent of conversion to A-type. The effect of Gbetagamma co-expression is occluded by treatment with dihydrocytochalasin B, a microfilament-disrupting agent shown previously by us to enhance the extent of conversion to A-type, and by overexpression of Kvbeta1.1. Gbeta1gamma2 subunits interact directly with GST fusion fragments of Kv1.1 and Kvbeta1.1. Co-expression of Gbeta1gamma2 causes co-immunoprecipitation with Kv1.1 of more Kvbeta1.1 subunits. Thus, we suggest that Gbeta1gamma2 directly affects the interaction between Kv1.1 and Kvbeta1.1 during channel assembly which, in turn, disrupts the ability of the channel to interact with microfilaments, resulting in an increased extent of A-type conversion.

Activation of a metabotropic glutamate receptor and protein kinase C reduce the extent of inactivation of the K+ channel Kv1.1/Kvbeta1.1 via dephosphorylation of Kv1.1.

Various brain K+ channels, which may normally exist as complexes of alpha (pore-forming) and beta (auxiliary) subunits, were subjected to regulation by metabotropic glutamate receptors. Kv1.1/Kvbeta1.1 is a voltage-dependent K+ channel composed of alpha and beta proteins that are widely expressed in the brain. Expression of this channel in Xenopus oocytes resulted in a current that had fast inactivating and noninactivating components. Previously we showed that basal and protein kinase A-induced phosphorylation of the alpha subunit at Ser-446 decreases the fraction of the noninactivating component. In this study we investigated the effect of protein kinase C (PKC) on the channel. We showed that a PKC-activating phorbol ester (phorbol 12-myristate 13-acetate (PMA)) increased the noninactivating fraction via activation of a PKC subtype that was inhibited by staurosporine and bisindolylmaleimide but not by calphostin C. However, it was not a PKC-induced phosphorylation but rather a dephosphorylation that mediated the effect. PMA reduced the basal phosphorylation of Ser-446 significantly in plasma membrane channels and failed to affect the inactivation of channels having an alpha subunit that was mutated at Ser-446. Also, the activation of coexpressed mGluR1a known to activate phospholipase C mimicked the effect of PMA on the inactivation via induction of dephosphorylation at Ser-446. Thus, this study identified a potential neuronal pathway initiated by activation of metabotropic glutamate receptor 1a coupled to a signaling cascade that possibly utilized PKC to induce dephosphorylation and thereby to decrease the extent of inactivation of a K+ channel.

Inactivation of a voltage-dependent K+ channel by beta subunit. Modulation by a phosphorylation-dependent interaction between the distal C terminus of alpha subunit and cytoskeleton.

Kv1.1/Kvbeta1.1 (alphabeta) K+ channel expressed in Xenopus oocytes was shown to have a fast inactivating current component. The fraction of this component (extent of inactivation) is increased by microfilament disruption induced by cytochalasins or by phosphorylation of the alpha subunit at Ser-446, which impairs the interaction of the channel with microfilaments. The relevant sites of interaction on the channel molecules have not been identified. Using a phosphorylation-deficient mutant of alpha, S446A, to ensure maximal basal interaction of the channel with the cytoskeleton, we show that one relevant site is the end of the C terminus of alpha. Truncation of the last six amino acids resulted in alphabeta channels with an extent of inactivation up to 2.5-fold larger and its further enhancement by cytochalasins being reduced 2-fold. The wild-type channels exhibited strong inactivation, which could not be markedly increased either by cytochalasins or by the C-terminal mutations, indicating that the interaction of the wild-type channels with microfilaments was minimal to begin with, presumably because of extensive basal phosphorylation. Since the C-terminal end of Kv1.1 was shown to participate in channel clustering via an interaction with members of the PSD-95 family of proteins, we propose that a similar interaction with an endogenous protein takes place, contributing to channel connection to the oocyte cytoskeleton. This is the first report to assign a modulatory role to such an interaction: together with the state of phosphorylation of the channel, it regulates the extent of inactivation conferred by the beta subunit.

Phosphorylation of a K+ channel alpha subunit modulates the inactivation conferred by a beta subunit. Involvement of cytoskeleton.

Voltage-gated K+ channels isolated from mammalian brain are composed of alpha and beta subunits. Interaction between coexpressed Kv1.1 (alpha) and Kvbeta1.1 (beta) subunits confers rapid inactivation on the delayed rectifier-type current that is observed when alpha subunits are expressed alone. Integrating electrophysiological and biochemical analyses, we show that the inactivation of the alphabeta current is not complete even when alpha is saturated with beta, and the alphabeta current has an inherent sustained component, indistinguishable from a pure alpha current. We further show that basal and protein kinase A-induced phosphorylations at Ser-446 of the alpha protein increase the extent, but not the rate, of inactivation of the alphabeta channel, without affecting the association between alpha and beta. In addition, the extent of inactivation is increased by agents that lead to microfilament depolymerization. The effects of phosphorylation and of microfilament depolymerization are not additive. Taken together, we suggest that phosphorylation, via a mechanism that involves the interaction of the alphabeta channel with microfilaments, enhances the extent of inactivation of the channel. Furthermore, phosphorylation at Ser-446 also increases current amplitudes of the alphabeta channel as was shown before for the alpha channel. Thus, phosphorylation enhances in concert inactivation and current amplitudes, thereby leading to a substantial increase in A-type activity.

Modulation by protein kinase C activation of rat brain delayed-rectifier K+ channel expressed in Xenopus oocytes.

The modulation by protein kinase C (PKC) of the RCK1 K+ channel was investigated in Xenopus oocytes by integration of two-electrode voltage clamp, site-directed mutagenesis and SDS-PAGE analysis techniques. Upon application of beta-phorbol 12-myristate 13-acetate (PMA) the current was inhibited by 50-90%. No changes in the voltage sensitivity of the channel, changes in membrane surface area or selective elimination of RCK1 protein from the plasma membrane could be detected. The inhibition was mimicked by 1-oleoyl-2-acetyl-rac-glycerol (OAG) but not by alphaPMA, and was blocked by staurosporine and calphostin C. Upon deletion of most of the N-terminus a preceding enhancement of about 40% of the current was prominent in response to PKC activation. Its physiological significance is discussed. The N-terminus deletion eliminated 50% of the inhibition. However, phosphorylation of none of the ten classical PKC phosphorylation sites on the channel molecule could account, by itself or in combination with others, for the inhibition. Thus, our results show that PKC activation can modulate the channel conductance in a bimodal fashion. The N-terminus is involved in the inhibition, however, not via its direct phosphorylation.

Regulation of RCK1 currents with a cAMP analog via enhanced protein synthesis and direct channel phosphorylation.

We have recently shown that the rat brain Kv1.1 (RCK1) voltage-gated K+ channel is partially phosphorylated in its basal state in Xenopus oocytes and can be further phosphorylated upon treatment for a short time with a cAMP analog (Ivanina, T., Perts, T., Thornhill, W. B., Levin, G., Dascal, N., and Lotan, I. (1994) Biochemistry 33, 8786-8792). In this study, we show, by two-electrode voltage clamp analysis, that whereas treatments for a short time with various cAMP analogs do not affect the channel function, prolonged treatment with 8-bromoadenosine 3',5'-cyclic monophosphorothioate ((Sp)-8-Br-cAMPS), a membrane-permeant cAMP analog, enhances the current amplitude. It also enhances the current amplitude through a mutant channel that cannot be phosphorylated by protein kinase A activation. The enhancement is inhibited in the presence of (Rp)-8-Br-cAMPS, a membrane-permeant protein kinase A inhibitor. Concomitant SDS-polyacrylamide gel electrophoresis analysis reveals that this treatment not only brings about phosphorylation of the wild-type channel, but also increases the amounts of both wild-type and mutant channel proteins; the latter effect can be inhibited by cycloheximide, a protein synthesis inhibitor. In the presence of cycloheximide, the (Sp)-8-Br-cAMPS treatment enhances only the wild-type current amplitudes and induces accumulation of wild-type channels in the plasma membrane of the oocyte. In summary, prolonged treatment with (Sp)-8-Br-cAMPS regulates RCK1 function via two pathways, a pathway leading to enhanced channel synthesis and a pathway involving channel phosphorylation that directs channels to the plasma membrane.

[Hemodynamic effects of intravenous allapinine in patients with myocardial infarction]. / Vliianie vnutrivennogo vvedeniia allapinina na gemodinamiku u bol'nykh infarktom miokarda.

Single intravenous allapinine, 30 mg, given to patients with acute myocardial infarction, including those with moderate circulatory insufficiency, fails to affect central hemodynamic parameters or has a favourable action: normalizes pulmonary diastolic pressure, cardiac index, diminishes total peripheral vascular resistance. The agent also produces a weak antihypertensive effect and increases heart rate.

[Evaluation of the overall contractile function of the right ventricle using two-dimensional echocardiography in patients with myocardial infarction]. / Otsenka obshchei sokratitel'noi funktsii pravogo zheludochka s pomoshch'iu dvukhmernoi ékhokardiografii u bol'nykh infarktom miokarda.

The paper presents two methods for calculating the contractile function of the right ventricle (RV): (1) three-plane one with the use of short- and long-axis-cut RV; (2) single-plane one which was modified to the use of long-axis RV images in the projection of 4 chambers. A total of 56 patients with first acute gross myocardial infarction were studied. A control group included 14 healthy subjects aged 25-60 years. The proposed three-plane method for estimating the volume parameters and ejection fraction of the right ventricle was shown to correlate with contrast ventriculography significantly. A high correlation was found between the stroke output of RV (as calculated for the left ventricle by the Chapman method) and that (by the three-plane method). The RV volume values in patients with anterior or inferior myocardial infarction suggest that there is a trend to suppress RV function in the patients, but it is more specific for those with inferior myocardial infarction.

[Effects of restored blood flow on the contractile function of the right ventricle in patients with acute myocardial infarct: data of two-dimensional computerized echocardiography]. / Vliianie vosstanovleniia krovotoka na sokratitel'nuiu funktsiiu pravogo zheludochka u bol'nykh ostrym infarktom miokarda po dannym dvukhmernoi kompiuternoi ékhokardiografii.

The volumes of the right and left ventricles were measured in 78 patients with first acute transmural myocardial infarction at days 1, 3, 7, 14, and 28 of the disease. Thirty four patients were diagnosed as having anterior myocardial infarction, 35 presented with inferior myocardial infarction, and 9 had a concurrent right and left ventricular inferior wall myocardial infarction. A high incidence of right ventricular dysfunction was confirmed both in anterior and inferior myocardial infarction. The most profound right ventricular contractile dysfunctions were detected in patients with proximal right coronary occlusion in the absence of reperfusion. Successful thrombolytic therapy for myocardial infarction was found to affect right ventricular function to a lesser extent than left function.

[Effect of thrombolytic, anticoagulant and anti-thrombocytic therapy on the development of left-ventricular thrombosis and the incidence of thromboembolic complications in patients with myocardial infarction]. / Vliianie tromboliticheskoi iantitrombotsitarnoi terapii na razvitie tromboza levogo zheludochka i chastoty tromboémbolicheskikh oslozh- nenii u bol'nykh infarktom miokarda.

To reveal the effects produced by thrombolytic and anticoagulative therapies on the formation of left ventricular mural thrombosis (LVT) and the frequency of thromboembolic events, a prospective randomized study was performed in 285 patients with primary transmural myocardial infarction. LVT was diagnosed from the serial two-dimensional echocardiographic findings. No significant effect of the therapy with thrombolytic and anticoagulative agents was found on the incidence and periods of intracavitary LVT development. There was a significant reduction in the incidence of LVT during early (less than 3 hours) reperfusion of the occluded coronary artery. Anticoagulative and antiplatelet therapies performed within the first month of myocardial infarction decrease the risk for systemic thromboembolism in patients with diagnosed LVT.

[Modern methods for the instrumental diagnosis of intracavitary thrombosis of the left ventricle in myocardial infarct]. / Sovremennye metody instrumental'noi diagnostiki vnutripolostnogo tromboza levogo zheludochka pri infarkte miokarda.

To study the sensitivity and specificity of two-dimensional echocardiography in the diagnosis of left ventricle thrombosis which often attends myocardial infarction and to define diagnostic potentialities of the new research methods (digital subtraction ventriculography and MR tomography), 449 patients with acute transmural myocardial infarction were examined. Based on the comparison of the echocardiographic readings and morbid anatomy data it has been shown that the sensitivity of two-dimensional echocardiography in revealing left ventricle thrombosis amounts to 89%, specificity to 88%. Digital subtraction ventriculography has a lower (77%) sensitivity, with the specificity being satisfactory enough (88%). Meanwhile MR tomography enables a highly precise diagnosis of left ventricle thrombosis and can be used as a supplementary diagnostic method in questionable cases and in thrombi small in size or in unsatisfactory ultrasonic visualization of the heart structures.

[The action of triftorfenazin on the Ca-ATPase activity of fibroblasts and Ehrlich ascites cells]. / Deistvie triftorfenazina na aktivnost' Ca-ATFazy fibroblastov i astsitnykh kletok Erlikha.

The authors studied the mechanisms underlying the increase of the Ca-ATPase activity in tumoral cells. The kinetic analysis showed that Michaelis constant for ATP was higher in comparison with proliferating cells.

[Postradiation changes in the activity of membrane-bound nerve tissue enzymes]. / Postradiatsionnye izmeneniia aktivnosti membranosviazannykh fermentov nervnoi tkani.

A study was made of the effect of various radiation doses (1.75 to 12.25 Gy) on the enzyme activity of Na,K-ATPase system of the microsomal brain fraction of mongrel and Wistar rats. With a similar method of the fraction isolation different response of the activity of this enzyme was registered. Different radiosensitivity of M9-ATPase is responsible for the direction of changes in the Na,K-ATPase activity of the preparations.

[The radiomodifying action of ATP and ADP on membrane-bound enzymes]. / Radiomodifitsiruiushchee deistvie ATF i ADF na membranosviazannye fermenty.

A modifying action of ATP and ADP on the activity of some key membrane-bound enzymes of the brain and heart microsomes of rats exposed to 7 Gy radiation has been investigated. The difference in the reactions of energy-dependent enzymes is attributed to the compensatory systems involved at the molecular level.

[Study of the effect of the restoration of coronary blood flow on left-ventricular contractility in patients with acute myocardial infarction based on the data of computerized two-dimensional echocardiography]. / Vliianie vosstanovleniia koronarnogo krovotoka na sokratitel'nuiu funktsiiu levogo zheludochka u bol'nykh ostrym infarktom miokarda po dannym komp'iuternoi dvukhmernoi ékhokardiografii.

Total and local left-ventricular contractility was assessed by computerized two-dimensional echocardiography in 52 patients with acute myocardial infarction. Three groups of patients were identified: those with recovered coronary flow (group 1), reperfusion failure (group 2) and the lack of occlusion at first coronarography (group 3). Patients from group 3 showed the most intact left-ventricular myocardial contractility and the most favorable clinical course of the disease, while second-group patients had particularly impaired left-ventricular contractility. In the first group, the size of the asynergic area diminished by day 28 of the disease to a greater extent, as compared to the second group. Therefore, coronary reperfusion within the first 6 hours after the attack shows correlation to a smaller asynergic zone and a more favorable clinical course of the disease.